Organogenesis of cartilage and bone

Organogenesis involves the formation of bone and cartilage as an organ (skeleton). Condensation of undifferentiated cells follows a pattern set by the early patterning genes. Differentiation of condensed cells into chondrocytes or osteoblasts follows, and these cells start producing a bone- or cartilage-specific extracellular matrix (a process referred to as histogenesis). These aspects of early skeletogenesis, the 'anlage', represent the outlines of the future skeletal elements. Bones then develop either directly from osteoblastic progenitor cells (desmal or intramembranous) or within a temporary framework of hyaline cartilage (endochondral). Recently, disease related genes have been characterized that are involved in the regulation of organogenesis. As expected, mutations in these genes result in altered shapes and/or numbers of certain skeletal elements since they affect the differentiation of progenitor cells and hence the formation of the anlage.

One example of such a gene is the recently identified transcription factor Cbfal. Cbfal belongs to the core binding factor (Cbf) transcription factors, a small family of heterodimeric proteins of two unrelated subunits comprising a DNA binding a subunit and a non-DNA binding fí subunit. The mammalian Cbfa subunits are encoded by three distinct genes, Cbfal, Cbfa2 and Cbfa3. They share a conserved 128 amino acid domain, called the runt domain because of its homology to the Drosophila pair-rule gene runt.

CBFA1 is mutated in cleidocranial dysplasia

CBFA1 is mutated in cleidocranial dysplasia (CCD), a dominantly inherited skeletal malformation syndrome (Mundlos et al 1997, Lee et al 1997, Quack et al 1999). Patients with CCD exhibit a number of typical clinical findings including (i) hypoplasia/aplasia of the clavicle, (ii) abnormal craniofacial growth, (iii) supernumerary teeth, (iv) short stature, and (v) several other minor skeletal changes (for review see Mundlos 1999) (Fig. 3).

Most mutations in CBFA1 are expected to result in heterozygous loss of function indicating that haploinsufficiency is sufficient to cause disease. Deletions ofthe entire gene, splice site mutations within the first exons, insertions resulting in frame shifts and missense mutations are common (Mundlos et al 1997). Two nonsense mutations located within the DNA binding runt domain were shown to interfere with DNA binding (Lee et al 1997). Other point mutations located in the region coding for the nuclear localization signal apparently inactivate this signal resulting in a protein that stays in the cytoplasm instead of being transported into the nucleus (Quack et al 1999).

Mutation analysis in a wide spectrum of individuals has helped to define and extend the phenotypic spectrum associated with mutations in CBFA1.

FIG. 3. Radiological findings in cleidocranial dysplasia. (A) Chest radiograph demonstrating cone-shaped thorax, low set shoulders and bilateral clavicular hypoplasia (right > left, arrows). (B) Panoramic radiograph of teeth from an affected patient aged 15 years showing multiple impacted teeth and partly persistent deciduous dentition.

FIG. 3. Radiological findings in cleidocranial dysplasia. (A) Chest radiograph demonstrating cone-shaped thorax, low set shoulders and bilateral clavicular hypoplasia (right > left, arrows). (B) Panoramic radiograph of teeth from an affected patient aged 15 years showing multiple impacted teeth and partly persistent deciduous dentition.

Supernumerary teeth may be the only finding, indicating that mutations in CBFA1 can be associated exclusively with a dental phenotype. One patient with a frame shift mutation 3' of the runt domain coding region had, in addition to severe CCD, congenital osteoporosis, severe skoliosis and recurrent fractures. At age 26 this patient had a bone density of — 6.34 (DEXA scan of left forearm, value in SD with <—1.5 representing osteopenia) (Quack et al 1999). These results indicate that CBFA1 has a role beyond development and is necessary for the

FIG. 4. Cbfal in skeletal development. Alizarin red alcian blue stained skeletal preparations of fore limbs from wild- type (+/+), Cbfa1+/- and Cbfa1-/- mice. Note the size of the clavicle (arrowed) in wild-type and heterozygous mice. The tuberositas humeri (circled) is absent in Cbfa1+/~ mice. The skeleton of Cbfa1~/~ mice is without signs of ossification and severely reduced in size. Note the defect in the scapula.

FIG. 4. Cbfal in skeletal development. Alizarin red alcian blue stained skeletal preparations of fore limbs from wild- type (+/+), Cbfa1+/- and Cbfa1-/- mice. Note the size of the clavicle (arrowed) in wild-type and heterozygous mice. The tuberositas humeri (circled) is absent in Cbfa1+/~ mice. The skeleton of Cbfa1~/~ mice is without signs of ossification and severely reduced in size. Note the defect in the scapula.

homeostasis of adult bone mass. This is in agreement with data generated from transgenic mice that express a dominant negative form of Cbfal under the control of an osteoblast-specific promotor (Ducy et al 1999).

The role of Cbfal in development has been elucidated by the generation of mutated mice in which the Cbfal gene locus was targeted (Otto et al 1997, Komori et al 1997). Inactivation of one allele results in skeletal changes that are remarkably similar to those observed in CCD (Fig. 4). Cbfal~/+ mice have hypoplastic clavicles and open fontanelles as well as other minor skeletal abnormalities frequently observed in CCD patients such as changes in the pelvis, vertebrae and ribs (Mundlos et al 1996, Otto et al 1997). The development of the clavicle has been investigated in these mice in some detail. The study by Huang et al (1997) demonstrates that the clavicle forms by the condensation ofmesenchyme as early as E13. Cells in the centre of the condensation differentiate into characteristic precursor cells that express markers of the chondrogenic (collagen type II) and osteoblastic (collagen type I) lineage. In Cbfal~/+ mice the condensation takes place, yet the differentiation into precursor cells and consequently into chondrocytes and osteoblasts is lacking. These studies suggested that CBFA1 is a crucial factor in skeletal development regulating the differentiation of mesenchymal stem cells in bone and cartilage precursors.

This concept is further substantiated by the phenotype of mice completely deficient for Cbfal. Such mice die immediately after birth owing to a complete absence of bone (Otto et al 1997, Komori et al 1997). Further histological analysis showed an arrest in endochondral, as well as membranous ossification. Cbfalmice develop normal cartilage anlagen but differentiation of mesenchymal stem cells into osteoblasts is completely missing (Fig. 4). Hence,

FIG. 5. Expression of Cbfal in mouse embryos of stage (A) E12.5, (B) E13.5 and (C) E14.5. Note strong expression in the primordia of the facial bones (A, B) and in the cartilaginous anlagen of radius and ulna (r,u), humerus (h), and in phalanges (p). Ossification appears in humerus and radius/ulna at E14.5. With permission from Kim et al (1999).

FIG. 5. Expression of Cbfal in mouse embryos of stage (A) E12.5, (B) E13.5 and (C) E14.5. Note strong expression in the primordia of the facial bones (A, B) and in the cartilaginous anlagen of radius and ulna (r,u), humerus (h), and in phalanges (p). Ossification appears in humerus and radius/ulna at E14.5. With permission from Kim et al (1999).

structures such as the skull, the mandible and the maxilla do not develop, whereas the cartilaginous anlagen are present but are not replaced by bone. Cbfal is thus essential for osteoblast differentiation.

More detailed investigations showed that Cbfal is not only essential for osteoblast formation, but also a major regulator of chondrocyte differentiation. In Cbfa1~/~ mice hypertrophy does not take place or is severely delayed (Kim et al 1999). Interestingly, the effect of Cbfal on chondrocyte differentiation differs in the three fore limb segments. Whereas the humerus completely lacks hypertrophic cells, radius and ulna are less affected and will eventually show some hypertrophy and ossification. In the phalanges, hypertrophy is initiated but not maintained. A direct role for Cbfal in chondrocyte differentiation is supported by its expression pattern. At E12.5 Cbfal is strongly expressed in all cartilage anlagen long before ossification is present (Fig. 5). Besides in the perichondrium and in mature osteoblasts Cbfal is expressed in hypertrophic and pre-hypertrophic chondrocytes. The expression in chondrocytes overlaps with that of collagen type X (exclusively expressed in hypertrophic cells) and that of indian hedgehog (expressed in pre-hypertrophic cells). A role for Cbfal in chondrocyte differentiation can provide an explanation for the short stature of CCD patients.

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